Immunogenic substances comprising a polyinosinic acid-polycytidilic acid based adjuvant

ABSTRACT

The present invention provides a polynucleotide adjuvant composition and methods of use in eliciting an immune response. The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition together with other immunogenic compositions such as an antigen (e.g., as in a vaccine). The present invention further contemplates methods of use of such adjuvant compositions, particularly in eliciting an immune response to an antigenic compound.

FIELD OF INVENTION

The invention generally relates to immunogenic compositions and methodsof their use. More specifically the invention relates to an immunogeniccomposition comprising a polynucleotide adjuvant in combination with oneor more antigenic substances to be used to elicit disease specificimmune response in a host.

BACKGROUND OF INVENTION

The immune system may exhibit both specific and nonspecific immunity.Nonspecific immunity encompasses various cells and mechanisms such asphagocytosis (the engulfing of foreign particles or antigens) bymacrophages or granulocytes, and natural killer (NK) cell activity,among others. Nonspecific immunity relies on mechanisms lessevolutionarily advanced and does not display the acquired nature ofspecificity and memory, which are exemplary hallmarks of a specificimmune response. The key differences between specific and nonspecificimmunity are based upon B and T cell specificity. These cellspredominantly acquire their responsiveness after activation with aspecific antigen and have mechanisms to display memory in the event offuture exposure to that specific antigen. As a result, vaccination(involving specificity and memory) is an effective protocol to protectagainst harmful pathogens.

Generally, B and T lymphocytes, which display specific receptors ontheir cell surface for a given antigen, produce specific immunity. Thespecific immune system may respond to different antigens in two ways: 1)humoral-mediated immunity, which includes B cell stimulation andproduction of antibodies or immunoglobulins, antigen and helper T cells(Th2), and 2) cell-mediated immunity, which generally involves T cellsincluding cytotoxic T lymphocytes (CTLs), although other cells are alsoinvolved in the generation of a CTL response (e.g., antigen presentingcells and Th1 cells).

In the continual pursuit for safer and more effective vaccines, newtechnologies, including recombinant, purification and synthetic methods,have been used to improve the quality and specificity of antigens used.Purified, sub-unit and synthesized antigens demonstrate increased safetybut diminished immunogenicity which has been one driver for theidentification of effective adjuvant. Thus an effective adjuvant isincreasingly an essential component of modern vaccines. Adjuvants aregenerally compounds, that when administered with an antigen (eithermixed with, or given prior to the administration of the antigen)enhances and/or modifies the immune response to that particular antigen.

Exemplary adjuvants that have been used to enhance an immune responseinclude aluminum compounds (all generally referred to as “Alum”),oil-in-water emulsions (complete Freund's adjuvant (CFA) is anoil-in-water emulsion containing dried, heat-killed Mycobacteriumtuberculosis organisms), Saponin (isolated from the bark of QuillajaSaponoria, the adjuvant active component known as Quile A), CpG ODN(synthetic oligodeoxynucleotide containing unmethylated CpGdinucleotides), monophosphoryl lipid A (MPL) derived from thelipopolysaccharide of Salmonella minnesota Re595, Liposomes (usuallymade up of biodegradable materials such as phospholipids) andbiodegradable polymer microspheres (made from a variety of polymers suchas, polyphosphazene and polyanhydrides). The adjuvant properties ofthese compounds have been evaluated with each adjuvant showingadvantages and disadvantages.

Polynucleotide complexes have been investigated for their variousapplications including acting as adjuvants. Double-stranded RNAs(dsRNAs) are very potent biologic modifiers that can exert a profoundinfluence on cells at nanomolar concentrations. The modulating effectsof dsRNA include a broad spectrum of actions at the molecular andcellular levels.

At the molecular level, dsRNAs can elicit biological effects such asinterferon synthesis, induction of protein kinase, enhancement ofhistocompatibility antigen and inhibition of metabolism. And at thecellular level, dsRNA can elicit biological effects such aspyrogenicity, mitogenicity, macrophage activation, activation of humoralimmunity, activation of cell-mediated immunity and induction ofantiviral state. Immunomodulating effects of dsRNAs has been disclosed.U.S. Pat. No. 4,124,702 disclosed that double stranded polynucleotidesinduced interferon induction in living animal cells. U.S. Pat. No.3,906,092 disclosed that the antibody response to an adjuvant typevaccine was augmented by incorporation in the vaccine of apolynucleotide or a complex of polynucleotides. Houston et al.established PICLC (polyinosinic acid polycytidylic acidpoly-L-lysinecarboxy-methylcellulose complex) as a potent adjuvant byincreasing primary antibody response without the aid of an additionaladjuvant.

Polyinosinic acid-polycytidylic acid (PIC), one of most studiedpolynucleotide complexes, was not effective when used in monkeys andhumans due to its instability in the body after administration. Thus,PIC has been modified in many ways to overcome one or anotherdeficiency. For example, a complex of polyriboinosinic-polyribocytidylicacid with poly-L-lysine hydrobromide is about 5 to 15 times as resistantto hydrolysis by pancreatic ribonuclease as the parent PIC.

Lin et al. described that an antiviral drug comprising polyinosinicpolycytidylic acid, kanamycin and calcium can be used as an adjuvant(Lin, et al., A new immunostimulatory complex (PICKCa) in experimentalrabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993;and Chinese Patent No. 93105862.7). The Chinese Patent No. 93105862.7provides for the use of the general composition of Poly I:C, kanamycinand calcium (PICKCa) as an adjuvant in a vaccine for human and mammalianapplication. However, Lin found that that the form of PICKCa originallyidentified does not provide the optimal efficacy/safety profile for useas an adjuvant and also induces unacceptable adverse side effects undercertain conditions.

The present invention provides novel immunogenic compositions thatexhibit improved safety and efficacy profiles; and methods of use ofsuch compositions. Subject immunogenic compositions include apolynucleotide adjuvant and an antigen.

LITERATURE

The following references may be of interest:

-   JP 1093540A2;-   U.S. Pat. No. 4,124,702-   U.S. Pat. No. 3,692,899-   U.S. Pat. No. 3,906,092-   U.S. Pat. No. 4,389,395-   U.S. Pat. No. 4,349,538-   U.S. Pat. No. 4,024,241-   U.S. Pat. No. 3,952,097-   Houston et al., Infection and Immunity, 14: 318-9, 1976C-   Wright and Adler-Moore, Biochemical and Biophysical Research    Communications, 131: 949-45, 1985-   Lin, et al., A new immunostimulatory complex (PICKCa) in    experimental rabies: antiviral and adjuvant effects, Arch Virol,    131: 307-19, 1993-   Chinese Patent 93105862.7-   Gupta R. K. et al., Adjuvants—a balance between toxicity and    adjuvanticity, Vaccine, 11:293-306, 1993-   Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca    Raton, Fla., 1987-   Sela, M., Science 166:1365-1374 (1969)-   U.S. Pat. No. 6,008,200-   Ellouz et al., Biochem. & Biophy. Res. Comm., 59:1317, 1974-   U.S. Pat. No. 4,094,971-   U.S. Pat. No. 4,101,536-   U.S. Pat. No. 4,153,684-   U.S. Pat. No. 4,235,771-   U.S. Pat. No. 4,323,559-   U.S. Pat. No. 4,327,085-   U.S. Pat. No. 4,185,089-   U.S. Pat. No. 4,082,736-   U.S. Pat. No. 4,369,178-   U.S. Pat. No. 4,314,998-   U.S. Pat. No. 4,082,735-   U.S. Pat. No. 4,186,194-   U.S. Pat. No. 6,468,558-   New Trends and Developments in Vaccines, edited by Voller et al.,    University Park Press, Baltimore, Md., USA, 1978-   Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston    (1997)-   Gupa R. K. and Siber G. R., Adjuvants for human vaccines—current    status, problems and future prospects, Vaccine, 13 (14): 1263-1276,    1995-   Richard T Kenney et al. Meeting Report—2^(nd) meeting on novel    adjuvants currently in/close to human clinical testing, Vaccine 20    2155-2163, 2002-   Laboratory Techniques in Rabies Edited by F X Meslin, M M Kaplan, H    Koprowski 4^(th) Edition ISBN 92 4 1544 1-   PCT Pat. CN2005/000810-   U.S. application Ser. No. 10/551,847. Filed Sep. 29, 2005.

SUMMARY OF THE INVENTION

The present invention relates to novel immunogenic compositionscomprising a polynucleotide adjuvant composition together with animmunogenic or antigenic substance, and methods of use in eliciting animmune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.1 is a graph showing an immune response to the administration ofa PIKA/HBsAg antigen composition.

FIG. 1.2 is a graph showing an immune response to the administration ofa PIKA/influenza antigen composition.

FIG. 1.3 a is a graph showing an immune response to the administrationof a PIKA/HIV antigen composition.

FIG. 1.3 b is a graph showing a percentage of CD4 positive cellsexpressing INF-gamma in response to the administration of a PIKA/HIVantigen composition.

FIG. 1.4 a is a graph showing an immune response to the administrationof a PIKA/anthrax antigen composition.

FIG. 1.4 b is a graph showing a percentage of CD4 positive cellsexpressing INF-gamma in response to the administration of a PIKA/anthraxantigen composition.

FIG. 1.5 a is a graph showing an immune response to the administrationof a PIKA/HSV composition.

FIG. 1.5 b is a graph showing a percentage of CD4 positive cellsexpressing INF-gamma in response to the administration of a PIKA/HSVantigen composition.

FIG. 1.6 a is a graph showing an immune response to the administrationof a PIKA/H5N1 composition.

FIG. 1.6 b is a graph showing a percentage of CD4 positive cellsexpressing INF-gamma in response to the administration of a PIKA/H5N1antigen composition.

FIG. 1.6 c is a graph showing a percentage of CD8 positive cellsexpressing INF-gamma in response to the administration of a PIKA/H5N1antigen composition.

FIG. 2 is a graph showing specific antibody levels after administrationof a PIKA/SARS composition.

FIG. 3 is a graph showing in-vivo specific antibody levels afteradministration of a PIKA/H5N1/H9N2 composition.

FIG. 4 is a graph showing comparative protection properties of threerabies vaccines.

FIGS. 5A-5E provide a table of exemplary viral pathogens which can serveas a source of antigen and diseases associated with these organisms.

FIGS. 6A-6D provide a table of exemplary bacterial pathogens which canserve as a source of antigen and diseases associated with theseorganisms.

FIGS. 7A-7B provide a table of exemplary fungal pathogens which canserve as a source of antigen and diseases associated with theseorganisms.

FIGS. 8A-8D provide a table of exemplary parasites which can serve as asource of antigen and diseases associated with these organisms.

FIGS. 9A-9C provide a table of exemplary cancers (e.g., by tissue type)which serve as a source of antigen.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of certain embodiments of the inventionand the Examples included herein. Throughout this application, wherepublications are referenced, the disclosures of these publications arehereby incorporated by reference, in their entireties, into thisapplication in order to describe more fully the state of art to whichthis invention pertains.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skilledin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “animmunogenic composition” includes a plurality of such compositions andreference to “the antigen” includes reference to one or more antigensand equivalents thereof known to those skilled in the art, and so forth.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

Definitions of Terms

Prior to setting forth details of the present invention it may be usefulto an understanding thereof to set forth definitions of several termsthat are used herein.

The term “adjuvant,” as used herein, refers to any substance or mixtureof substances that increases or diversifies the immune response of ahost to an antigenic compound. Specifically:

-   -   1. The term “PICKCa” generally refers to a composition of poly        I:C, kanamycin and calcium irrespective of particular physical        and immunogenic properties.    -   2. “Av-PICKCa” refers to a form of PICKCa used commercially as        an antiviral drug.    -   3. “PIKA” refers to a composition of the invention comprising        poly I:C, an antibiotic (e.g., kanamycin), and a positive ion        (e.g., calcium), where the PIKA is characterized by physical        characteristics (e.g., molecular weight, size, and the like)        such that upon administration, PIKA exhibits characteristics of        an adjuvant with reduced adverse side effects (e.g., reduced        toxicity) relative to, for example, PICKCa and greater potency        (e.g., stimulates an enhanced immune response) relative to, for        example, Av-PICKCa.

The term “Poly I:C” or “PIC” refers to a composition containingpolyriboinosinic and polyribocytidylic nucleic acids, which may also bereferred to as polyinosinic acid-polycytidylic acid, respectively.

“PIC-containing molecule” or “PIC-containing compound” refers to,without limitation, PIC, which may be optionally complexed or otherwisecombined with at least one or both of an antibiotic (e.g., kanamycin)and a positive ion (e.g., calcium) present in a composition containingthe PIC-containing molecule. In one embodiment, the PIC-containingmolecule does not include poly-L-lysine or a derivative thereof in thecomplex.

“Heterogeneous” as used herein in the context of the adjuvantcompositions of the invention indicates that components of thecomposition, e.g., the PIC-containing molecules, are not uniform withrespect to a physical characteristic of molecular weight, size, or both.Where a composition is described as heterogenous for a given physicalcharacteristic, and is further described by a range of values for thatphysical characteristic, the composition is said to be composedsubstantially of molecules characterized by molecules having a physicalcharacteristic that is distributed within and across the recited range.While the composition may not contain a molecule representative of everyphysical characteristic value within the upper and lower limits of arecited range, the composition will generally include at least onemolecule having the physical characteristic of the upper value and ofthe lower value. The composition in certain embodiments may includemolecules outside the stated range of physical characteristics used todescribe the composition. The molecules that are present in thecomposition outside the prescribed range do not materially affect thebasic and novel characteristics of the composition.

The term “individual,” used interchangeably herein with “host,”“subject,” and “animal,” includes humans and all domestic, e.g livestockand pets, and wild mammals and fowl, including, without limitation,cattle, horses, cows, swine, sheep, goats, dogs, cats, rabbits, deer,mink, chickens, ducks, geese, turkeys, game hens, and the like.

The term “antibody” includes polyclonal and monoclonal antibodies, aswell as antigenic compound binding fragments of such antibodiesincluding Fab, F(ab′)2, Fd, Fv fragments, and single chain derivativesof the same. In addition, the term “antibody” includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, chimeric, bifunctional and humanized antibodies,and related synthetic isoforms. The term “antibody” is usedinterchangeably with “immunoglobulin.”

As used herein, the term “antigenic compound” refers to any substancethat can be recognized by the immune system (e.g., bound by an antibodyor processed so as to elicit a cellular immune response) underappropriate conditions.

An “antigen” as used herein includes but is not limited to cells; cellextracts; proteins; lipoproteins; glycoproteins; nucleoproteins;polypeptides; peptides; polysaccharides; polysaccharide conjugates;peptide mimics of polysaccharides; lipids; glycolipids; carbohydrates;viruses; viral extracts; multicellular organisms such as parasites; andallergens. Antigens may be exogenous (e.g., from a source other than theindividual to whom the antigen is administered, e.g., from a differentspecies) or endogenous (e.g., originating from within the host, e.g., adiseased element of body, a cancer antigen, a virus infected cellproducing antigen, and the like). Antigens may be native (e.g.,naturally-occurring); synthetic; or recombinant. Antigens include crudeextracts; whole cells; and purified antigens, where “purified” indicatesthat the antigen is in a form that is enriched relative to theenvironment in which the antigen normally occurs and/or relative to thecrude extract, for example, a cultured form of the antigen.

An “immunogenic composition” as used here in refers to a combination oftwo or more substances (e.g., an antigen and an adjuvant) that togetherelicit an immune response when administered to a host.

The term “polypeptide,” “peptide,” “oligopeptide,” and “protein,” areused interchangeably herein, and refer to a polymeric form of aminoacids of any length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

An “effective amount of an antigenic compound” refers to an amount ofantigenic compound which, in optional combination with an adjuvant, willcause the subject to produce a specific immunological response to theantigenic compound.

The term “immune response” refers to any response to an antigenic orimmunogenic compound by the immune system of a vertebrate subject.Exemplary immune responses include, but are not limited to local andsystemic cellular as well as humoral immunity, such as cytotoxic Tlymphocytes (CTL) responses, including antigen-specific induction ofCD8+ CTLs, helper T-cell responses including T-cell proliferativeresponses and cytokine release, and B-cell responses including antibodyresponse.

The term “eliciting an immune response” is used herein generally toencompass induction and/or potentiation of an immune response.

The term “inducing an immune response” refers to an immune response thatis stimulated, initiated, or induced.

The term “potentiating an immune response” refers to a pre-existingimmune response that is improved, furthered, supplemented, amplified,enhanced, increased or prolonged.

The expression “enhanced immune response” or similar means that theimmune response is elevated, improved or enhanced to the benefit of thehost relative to the prior immune response status, for example, beforethe administration of an immunogenic composition of the invention.

The terms “humoral immunity” and “humoral immune response” refer to theform of immunity in which antibody molecules are produced in response toantigenic stimulation.

The terms “cell-mediated immunity” and “cell-mediated immune response”are meant to refer to the immunological defense provided by lymphocytes,such as that defense provided by T cell lymphocytes when they come intoclose proximity to their victim cells. A cell-mediated immune responsenormally includes lymphocyte proliferation. When “lymphocyteproliferation” is measured, the ability of lymphocytes to proliferate inresponse to a specific antigen is measured. Lymphocyte proliferation ismeant to refer to B cell, T-helper cell or cytotoxic T-lymphocyte (CTL)cell proliferation.

The term “immunogenic amount” refers to an amount of antigenic compoundsufficient to stimulate an immune response, when administered with asubject immunogenic composition, as compared with the immune responseelicited by the antigen in the absence of the polynucleotide adjuvant.

The term “immunopotentiating amount” refers to the amount of theadjuvant needed to effect an increase in antibody titer and/orcell-mediated immunity when administered with an antigenic compound in acomposition of the invention, as compared with the increase in antibodyand/or cell mediated immunity level observed in the absence of thepolynucleotide adjuvant.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in asubject, particularly a mammalian subject, more particularly a human,and includes: (a) preventing the disease or symptom from occurring in asubject which may be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,e.g., arresting its development; or relieving the disease symptom, i.e.,causing regression of the disease or symptom (c) reduction of a level ofa product produced by the infectious agent of a disease (e.g., a toxin,an antigen, and the like); and (d) reducing an undesired physiologicalresponse to the infectious agent of a disease (e.g., fever, tissueedema, and the like).

As used herein, the term “mixing” includes any method to combine thecomponents of the composition; such methods include, but are not limitedto, blending, dispensing, dissolving, emulsifying, coagulating,suspending, or otherwise physically combining the components of thecomposition.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of compounds of the presentinvention calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically/physiologically acceptablediluent, carrier or vehicle.

EXEMPLARY EMBODIMENTS OF THE INVENTION

The present invention is directed to immunogenic compositions andmethods useful for the induction and/or enhancement of an immuneresponse, which may be humoral and/or cell-mediated, in a human, anon-human animal, or cell culture. In general, a subject immunogeniccomposition comprises an antigen (an “antigenic composition”) anadjuvant. The presence of the adjuvant enhances or modifies the immuneresponse to the antigen. The adjuvant may alter the quality of theimmune response by affecting the subclasses (isotypes) ofimmunoglobulins, chemokines, and/or cytokines produced. As a result theinnate immunity, humoral and/or cell-mediated immune responses are moreeffective with the presence of the adjuvant.

A particular advantage is the effectiveness of the PIKA adjuvant incombination with an antigenic substance in inducing a specific humoralimmune response thereby enhancing protective immunity.

A further important advantage is that the PIKA adjuvant in combinationwith an antigen can induce a specific cell mediated immune response thatis essential for a therapeutic vaccine for limiting and treatingintracellular viral, bacterial and parasite infections as well as forchronic diseases therapies such the treatment of cancers.

Accordingly, included in the invention are compositions having theunique product attributes that make them most suitable for use asvaccines to be administered to animals and/or humans that address theneed for a safe adjuvant, which elicits the maximum desired immuneresponse.

Accordingly, the present invention provides an adjuvant and immunogeniccompositions that can be used safely in humans and animals.

In some embodiments, a PIKA adjuvant composition comprising apolynucleotide, an antibiotic and a positive ion, wherein thepolynucleotide may be polyriboinosinic-polyribocytidylic acid (PIC); theantibiotic may be kanamycin, and the ion may be calcium.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting an antigen specific cell mediated immune response.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting an antigen specific humoral immune response.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting a combined specific cell and humoral immuneresponse

In one aspect of particular interest, the invention provides for anadjuvant composition or immunogenic composition comprising an adjuvantcomposition wherein the adjuvant composition or the immunogeniccomposition is freeze-dried.

In one aspect of particular interest, the invention provides for the useof a polynucleotide adjuvant composition for the preparation of amedicament for enhancing the immunogenic response of a host.

Polynucleotide Adjuvant

A subject immunogenic composition comprises a PIC-containingpolynucleotide adjuvant, e.g., a PIKA composition, is generally composedof polyinosinic acid, polycytidylic acid, an antibiotic (e.g.,kanamycin), and a divalent cation (e.g., calcium). It will be understoodthat reference to PIKA herein is exemplary of such PIC-containingadjuvants.

PIC-containing adjuvants of interest can be manufactured using methodsavailable in the art. The PIC-containing adjuvant composition can bemanufactured through any appropriate process. For example thepolynucleotide adjuvant composition can be manufactured by mixing ofpolyinosinic acid, polycytidylic acid, an antibiotic and the source of apositive ion in a sodium chloride/phosphate buffer solution that has apH between pH6 and pH8. The polyinosinic acid and polycytidylic acid aregenerally provided at a concentration of 0.1 to 10 mg/ml, usually 0.5 to5 mg/ml and more usually 0.5 to 2.5 mg/ml. The hyperchromicity valueshould be greater than 10%, greater than 15%, greater than 20%, orgreater than 50%. The preparation of the PIC and the combination withthe antibiotic (e.g., kanamycin) and the positive ion (e.g., calcium) isgenerally conducted under quality standards consistent withinternational Good Manufacturing Process.

In certain embodiments of the present invention, the antibioticcomponent of the adjuvant is kanamycin. Where the antibiotic iskanamycin, in some embodiments, the kanamycin in the polynucleotideadjuvant composition is used together with or substituted by one or moreantibiotics selected from the group including tobramycin,anthracyclines, butirosin sulfate, gentamicins, hygromycin, amikacin,dibekacin, nebramycin, metrzamide, neomycin, puromycin, streptomycin andstreptozocin. The antibiotic (e.g., Kanamycin or the like) in thepolynucleotide adjuvant composition of the invention is generallyprovided at a concentration of from about 10 units/ml to 100,000units/ml, from about 100 units/ml to 10,000 units/ml, or from about 500units/ml to 5,000 units/ml.

In certain embodiments of the present invention, the polynucleotideadjuvant composition further comprises a positive ion (cation), usuallya divalent cation, normally a cation of an alkali metal. The positiveion is generally provided in the composition of the invention as asource of positive ions such as a salt or complex, e.g., an organic orinorganic salt or complex, usually an inorganic salt or organic complex.Exemplary positive ions include, but are not necessarily limited to,calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt,deuterium, gallium, iodine, iron, or zinc.

The positive ion can be provided in the form of any suitable salt ororganic complex, including, but not necessarily limited to chloride,fluoride, hydroxide, phosphate, or sulfate salts. For example, where thepositive ion is calcium, the ion can be in the form of calciumcarbonate, calcium chloride, calcium fluoride, calcium hydroxide,calcium phosphates, or calcium sulfate.

The positive ion (e.g. calcium) can be provided in the composition ofthe invention at a concentration in the range of from about 10 umol to10 mmol/ml, usually from about 50 umol to 5 mmol/ml, and more usuallyfrom about 100 umol to 1 mmol/ml. The term “umol” is used throughout torefer to micromole.

Where the positive ion in the adjuvant composition of the invention iscalcium, it can be in combination with or substituted by other positiveions, including cadmium, lithium, magnesium, cerium, cesium, chromium,cobalt, deuterium, gallium, iodine, iron, and zinc, wherein the ions canbe in the form of inorganic salts or organic complexes.

The resulting composition is a PIC-containing adjuvant that furthercontains an antibiotic and a positive ion. In a particular embodiment,where the antibiotic is kanamycin and the ion is calcium the product maybe described as PICKCa. In a related embodiment the PICKCa compositionmay contain molecules without restriction of different physicalcharacteristics.

PIKA Adjuvant Composition

In a particular exemplary embodiments, the PIC-containing adjuvant isPIKA. PIKA may be produced in a variety of ways, with production fromPICKCa being of particular interest. PIKA can be produced from PICKCathrough additional manufacturing processes that involve the isolationand/or concentration of molecules of a defined molecular size and/orweight. The separation and concentration of polynucleotide molecules ofparticular characteristics using filtration, chromatography, thermaltreatment, centrifugal separation, electrophoresis, and similar methodsthat are standard processes and are known to those skilled in the art.

In embodiments of particular interest, the invention features anadjuvant generally referred to as PIKA comprising apolyriboinosinic-polyribocytidylic acid (PIC), an antibiotic (e.g.,kanamycin), and a positively charged ion (e.g., a calcium ion), whereinthe composition contains molecules of the adjuvant heterogeneous formolecular weight having a molecular weight of from about 66,000 to1,200,000 Daltons. That is, the adjuvant composition comprises moleculeswith a weight distribution in the range of from about 66,000 to1,200,000 Daltons.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition are heterogeneous, that is the weight ofthe adjuvant molecules are distributed within a range of molecularweight, where the molecular weight is from about 300,000 to 1,200,000Daltons, or from about 66,000 to 660,000 Daltons, or from about 300,000to 660,000 Daltons, or from about 300,000 to 2,000,000 Daltons, or fromabout 66,000 Daltons to about 100,000 Daltons, 100,000 to 200,000Daltons, from about 300,000 Daltons to about 4,000,000 Daltons, or fromabout 500,000 Daltons to 1,000,000 Daltons, or from about 1,000,000Daltons to 1,500,000 Daltons, or from about 1,500,000 Daltons to2,000,000 Daltons, or from about 2,000,000 Daltons to 2,500,000 Daltons,or from about 2,500,000 Daltons to 3,000,000 Daltons, or from about3,000,000 Daltons to 3,500,000 Daltons, or from about 3,500,000 Daltonsto 4,000,000 Daltons, or from about 4,000,000 Daltons to 4,500,000Daltons, or from about 4,500,000 Daltons to 5,000,000 Daltons.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition have an average molecular weight equal orequal to or greater than 150,000 Daltons, or equal to or greater than250,000 Daltons, or equal to or greater than 350,000 Daltons, or equalto or greater than 500,000 Daltons, or equal to or greater than 650,000Daltons, or equal to or greater than 750,000 Daltons, or equal to orgreater than 1,000,000 Daltons, or equal to or greater than 1,200,000Daltons, or equal to or greater than 1,500,000 Daltons, or equal to orgreater than 2,000,000 Daltons.

In embodiments of particular interest, the invention features anadjuvant generally referred to as PIKA comprising apolyriboinosinic-polyribocytidylic acid (PIC), an antibiotic, and apositive ion wherein the composition contains molecules of the adjuvantheterogeneous, that is the size of the adjuvant molecules aredistributed within a range of molecular size, for molecular size havinga sediment co-efficient Svedbergs (S) of from about 6.43 S to 24.03 S.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition are heterogeneous, that is the size of theadjuvant molecules are distributed within a range of molecular size,where the molecular size is from about 12.8 S to 24.03 S, or from about3 S to 12 S or from about 6.43 to 18.31 S, or from about 12.8 to 18.31S, or from about 12.8 S to 30.31 S, or from about 12.8 S to 41.54 S, orfrom about 13.5 S, to 18.31 S, or from about 13.5 S to 24.03 S, or fromabout 16.14 to 22.12 S, or from about 22.12 S to 26.6 S, or from about26.6 S to 30.31 S, or from about 30.31 S to 33.55 S, or from about 33.55S to 36.45 S, or from about 36.45 S to 39.1 S, or from about 39.1 S to41.54 S, or from about 41.54 S to 43.83 S, or from about 43.83 S to45.95 S.

In further related embodiments, the PIKA polynucleotide adjuvantcomposition has an average sedimentation co-efficient (Svedbergs)greater than 9, or greater than 12, or greater than 13.5, or greaterthan 15, or greater than 16, or greater than 17, or greater than 18, orgreater than 19, or greater than 20, or greater than 21, or greater than22 or greater than 25, or greater than 30.

Immunogenic Properties

An immunogenic composition, including PIKA and an antigen, can generallyinduce an antigen-specific immune response in at least two ways: i)humoral-mediated immunity, which includes B cell stimulation andproduction of antibodies or immunoglobulins (other cells are alsoinvolved in the generation of an antibody response, e.g.antigen-presenting cells, including macrophages and helper T cells (Th1and Th2), and ii) cell-mediated immunity, which generally involves Tcells including cytotoxic T lymphocytes, although other cells are alsoinvolved in the generation of a cytotoxic T lymphocyte response (e.g.,Th1 and/or Th2 cells and antigen presenting cells). Furthermore, thepolynucleotide adjuvant composition may alter the quality of the immuneresponse by affecting the subclasses (isotypes) of immunoglobulinsproduced, as well as their affinities.

The degree and nature of the immunogenic response induced by a subjectimmunogenic composition may be thus assessed by measuring the presenceof molecules including cytokines, chemokines and antibodies produced bycells of the immune system.

Interleukin-4 is mainly produced by activated Th2 cells. The productionof Interlukin-4 (IL-4) induces the activation of B cells and thereby theproduction of IgG1 and IgE immunoglobulins (antibodies) which may bemeasured in the samples of blood serum. IL-4 is considered as anindictor and typical cytokine of Th2 immune response. Th2 cells tend topromote antibody response.

Interleukin-2 (IL-2) is mainly produced by activated Th1 cell as well asNK and lymphokine-activated killer (LAK) cells. IL-2 is instrumental inthe proliferation and maturing of T cells an essential stage in aneffective cell mediated adaptive immune response.

Interferon-γ (INF-γ), which may be produced by a variety of cellsincluding natural killer cells as well as both CD4⁺ and CD8⁺ T cells,plays an essential part in the adaptive immune response including theactivation of macrophages to become highly microbicidal. Further, INF-γis an influencing factor in directing the development of specificallyTh1 T cells thereby up-regulating a cell mediated adaptive immuneresponse.

The invention contemplates methods of use of the polynucleotide adjuvantof the invention with an antigen, for example, to elicit an antigenspecific humoral response and/or specific cellular (e.g., T cell)response in a subject. The immune response elicited may be a response toan antigen in a naïve subject, or may serve to enhance an existingimmune response (e.g., as in a booster). It has been found thatimmunogenic compositions according to the invention comprising PIKA haveparticularly advantageous properties as described herein.

A variety of different antigens were tested in vivo for their ability toinduce an immune response with and without the PIKA adjuvant. Theantigens tested include: a recombinant protein hepatitis B surfaceantigen type adw, an inactivated split influenza vaccine (VAXIGRIP fromSanofi Pasteur), a synthesized HIV peptide antigen, a recombinantprotein herpes simplex virus type 2 gD antigen, recombinant protectiveanthrax protein antigen, inactivated whole virus avian influenza antigenstrain H5N1 and an inactivated whole virus Severe Acute RespiratorySyndrome (SARS) inactivated antigen.

In each case presence of the PIKA adjuvant together with the antigenenhanced the expression of cytokines when compared with the antigen orPIKA alone. In particular the enhanced expressions of the cytokinesINF-γ, IL-2 and IL-4 (see Examples/FIGS. 1.1, 1.2, 1.3, 1.4, 1.5 and1.6) indicates the stimulation of a specific adaptive immunity wasgreater with the presence of the PIKA adjuvant and more specifically theenhanced expressions of the cytokines INF-γ, IL-2 indicates thepredominant Th1 cell immunity was significantly improved with thepresence of the PIKA adjuvant. The activity of a cell mediated immuneresponse is a key feature essential for treating intracellular viral,bacterial and parasite infection and particularly important factor fordeveloping a therapeutic vaccine.

Further the composition containing PIKA stimulated INF-γ production byCD4+ T cells (Examples/FIGS. 1.3, 1.4, 1.5 and 1.6) and CD8+ T cells(Example/FIG. 1.6). This feature validates that PIKA is enhancing theadaptive immune response and in particular up-regulating the cellmediated immunity of the host.

The observed proliferation of antibodies in the blood serum (seeExamples/FIGS. 2 and 3) demonstrates that the PIKA adjuvant induces abeneficial humoral response.

The PIKA adjuvant enhances the immune response in a host when combinedwith a inactivated antigen (Example/FIGS. 1.2, 1.6 and Examples/FIGS. 2,3 and 4), a peptide antigen (Example/FIG. 1.3) and a recombinant antigen(Examples/FIGS. 1.1, 1.4, 1.5).

A particular feature of the PIKA adjuvant is to provide adequateprotection to both limit and/or eradicate infection in a host, and/or toreduce the risk of symptoms of a disease that could result frominfection by a pathogen. VAXIGRIP (Sanofi Pasteur) used as an antigen inExample/Figure 1.2 is itself a human influenza vaccine that elicits adegree of immune activity considered sufficient to provide protectionagainst an actual influenza infection. The addition of PIKA to VAXIGRIPfurther enhanced the immune response as demonstrated by the degree ofbeneficial cytokines (IL-2, INF-γ and IL-4) expressed by the immunesystem.

In a further demonstration of PIKA's protective properties, 24 ten-dayold chickens were inoculated with a composition comprising PIKA andinactivated avian influenza antigens including strains H5N1 and H9N2(Example/FIG. 3). The chickens were subsequently challenged with thelive H5N1 virus and observed for a two week period. At the end of theprogram the survival rate for the chickens inoculated with thePIKA/antigen composition was 83% as compared with only 17% for a controlgroup of 24 chickens that were exposed to the live virus without priorinoculation with the PIKA/antigen composition.

In a related experiment to demonstrate the therapeutic enhancingproperties of the PIKA adjuvant, Balb/c mice were challenged with astrain of wild rabies virus (Example/FIG. 4). Post infection the threedifferent groups of animals were inoculated with a regime of treatmentswith different rabies vaccines. The survival rate of the group of miceinoculated with a combination of an inactivated purified hamster kidneycell rabies antigen plus PIKA attained 80%. The survival rate of thesecond group of mice administered with a hamster kidney cell purifiedrabies antigen with an alum adjuvant was 15%. Further the third group ofmice administered with the Sanofi-Aventis' “Verorab”—vero cellinactivated rabies vaccine had a survival rate of 10%.

Additional Features

In a further embodiment, a subject immunogenic composition is furtherdefined by the relative presence of the PIKA adjuvant and the antigen orantigens where the presence is measured in terms of one or morecharacteristics of quantity, concentration, volume, number of moleculesor other recognized metric.

In related embodiments, a subject immunogenic composition comprises apolynucleotide adjuvant composition and an antigen or antigens where thepresence of the adjuvant and the antigen in terms of weight or number ofmolecules is in a ratio of less than 1 to 1,000, of less than 1 to 900,of less than 1 to 800, of less than 1 to 700, of less than 1 to 500, ofless than 1 to 400, of less than 1 to 300, of less than 1 to 200, ofless than 1 to 100, of less than 1 to 50, of less than 1 to 10, of lessthan 1 to 5, of less than 1 to 2, of about 1 to 1, of greater than 2 to1, of greater than 5 to 1, of greater than 10 to 1, of greater than 50to 1, of greater than 100 to 1, of greater than 200 to 1, of greaterthan 300 to 1, of greater than 400 to 1, of greater than 500 to 1, ofgreater than 600 to 1, of greater than 700 to 1, of greater than 800 to1, of greater than 900 to 1, of greater than 1,000 to 1

In a further related embodiment a subject immunogenic composition isdefined in terms of dose; that is the quantity of vaccine that is to beadministered to induce the optimal beneficial immune response oralternatively the range of dose that may be administered from theminimum required to elicit an immune response to the maximum dose beyondwhich the incremental beneficial response is not medically justified inthe context of the potential inducement of adverse side effects.

In certain embodiments of particular interest the immunogeniccomposition comprises the polynucleotide adjuvant composition andantigen where the presence of the antigen in a unit dose is provided ina quantity, that is more than 0.001 mg is more than 0.005 mg, is morethan 0.01 mg, is more than 0.025 mg, is more than 0.05 mg, is more than0.075 mg, 0.1 mg is more than 0.25 mg, is more than 0.5 mg, is more than1.2 mg, is more than 1.4 mg, is more than 1.6 mg, is more than 1.8 mg,is more than 2.0 mg is more than 2.5 mg, is more than 3 mg, is more than3.5 mg, is more than 4 mg, is more than 5 mg, is more than 6 mg, is morethan 7 mg, is more than 8 mg, is more than 9 mg, is more than 10 mg, ismore than 15 mg, is more than 20 mg, is more than 25 mg, or is more than50 mg.

An optimal amount of antigen and the optimal ratio of antigen to PIKAadjuvant can be ascertained by standard studies involving observationsof antibody titers and other immunogenic responses in the host.

Antigens

In an embodiment of particular interest the invention provides for apolynucleotide adjuvant composition together with an antigen or vaccinewhere the source of the antigen is a human antigen, a animal antigen, aplant antigen, one or more agents from infectious agents from any virus,bacteria including mycobacterium, fungus or parasite, cancer antigen,allergenic agents and other antigens, such as for developing autoimmunediseases.

In certain embodiments, the antigens may be derived from a naturalsource either crude or purified and used in its original live form orafter having been killed, or inactivated, or truncated, or attenuated,or transformed into a nonreverting form, or detoxified, or mutated intoa nontoxic form, or filtered or purified.

In some embodiments, the antigen is an isolated micro-organism antigenfor example, a viral antigen, a bacterial antigen, a fungal antigen, anallergy antigen, a cancer antigen or an autoimmune antigen. In otherembodiments, the antigen is a whole, inactivated antigen. Methods ofinactivating a whole antigens are well known in the art; any knownmethod can be used to inactivate an antigen and can be selectedappropriately for the type of antigen of interest. Such methods ofinactivating an antigen include for example, use of photoreactivecompounds; oxidizing agents; irradiation (e.g., UV irradiation;γ-irradiation); combinations of riboflavin and UV irradiation;solvent-detergent treatment (e.g., treatment with organic solventtri-N-butyl-phosphate with a detergent such as Tween 80); polyethyleneglycol treatment; pasteurization (heat treatment); and low pH treatment;mild enzymatic treatment with pepsin or trypsin; Methylene blue (MB)phototreatment; treatment with Dimethylmethylene blue (DMMB) and visiblelight; treatment with S-59, a psoralen derivative and UVA illumination;and the like. In a related embodiment of particular interest the antigenmay be synthesized by means of solid phase synthesis, or may be obtainedby means of recombinant genetics, or may be otherwise manufacturedartificially so as to imitate the immunogenic properties of a pathogen.

Polypeptide antigens may be isolated from natural sources using standardmethods of protein purification known in the art, including, but notlimited to, liquid chromatography (e.g., high performance liquidchromatography, fast protein liquid chromatography, etc.), sizeexclusion chromatography, gel electrophoresis (including one-dimensionalgel electrophoresis, two-dimensional gel electrophoresis), affinitychromatography, or other purification technique. One may employ solidphase peptide synthesis techniques, where such techniques are known tothose of skill in the art. See Jones, The Chemical Synthesis of Peptides(Clarendon Press, Oxford)(1994). Generally, in such methods a peptide isproduced through the sequential additional of activated monomeric unitsto a solid phase bound growing peptide chain. Well-establishedrecombinant DNA techniques can be employed for production ofpolypeptides, where, e.g., an expression construct comprising anucleotide sequence encoding a polypeptide is introduced into anappropriate host cell (e.g., a eukaryotic host cell grown as aunicellular entity in in vitro cell culture, e.g., a yeast cell, aninsect cell, a mammalian cell, etc.) or a prokaryotic cell (e.g., grownin in vitro cell culture), generating a genetically modified host cell;under appropriate culture conditions, the protein is produced by thegenetically modified host cell.

In some embodiments, the antigen is a purified antigen, e.g., from about25% to 50% pure, from about 50% to about 75% pure, from about 75% toabout 85% pure, from about 85% to about 90% pure, from about 90% toabout 95% pure, from about 95% to about 98% pure, from about 98% toabout 99% pure, or greater than 99% pure.

The antigen may be a cellular, capsular, infectious clone, replicon,vectored, microencapsulated, monovalent, bivalent or multivalent.

The polynucleotide adjuvant composition of the present invention canalso be utilized to enhance the immune response against antigensproduced by the use of DNA vaccines and/or DNA expressed proteins. TheDNA sequences in these vaccines coding for the antigen can be either“naked” or contained in a delivery system, such as liposomes.

In one aspect of particular interest a subject immunogenic compositionmay be defined by the selection of antigen or antigens that are used incombination with the PIKA adjuvant.

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a viral antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the viruses described in FIGS. 5A-5E.

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a bacterial antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the bacteria described in FIGS. 6A-6D

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a fungal antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the fungi described in FIGS. 7A-7B.

More specifically, the present invention provides for an immunogeniccomposition and method of use, where the immunogenic compositioncomprises a PIKA adjuvant together with a parasitic antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the parasites described in FIGS. 8A-8D.

In a related embodiment, the present invention provides for animmunogenic composition and method of use, where the immunogeniccomposition comprises a PIKA adjuvant together with an allergy antigen(“allergen”) or vaccine where the source of the antigen or vaccine isderived from or produced to emulate a pathogen from a human or animalallergy sources including; plants, animals, fungi, insects, food, drugs,dust, and mites and the like.

Allergens include but are not limited to environmental aeroallergens;plant pollens such as ragweed/hayfever; weed pollen allergens; grasspollen allergens; Johnson grass; tree pollen allergens; ryegrass;arachnid allergens, such as house dust mite allergens (e.g., Der p I,Der f I, etc.); storage mite allergens; Japanese cedar pollen/hay fever;mold spore allergens; animal allergens (e.g., dog, guinea pig, hamster,gerbil, rat, mouse, etc., allergens); food allergens (e.g., allergens ofcrustaceans; nuts, such as peanuts; citrus fruits); insect allergens;venoms: (Hymenoptera, yellow jacket, honey bee, wasp, hornet, fire ant);Other environmental insect allergens from cockroaches, fleas,mosquitoes, etc.; bacterial allergens such as streptococcal antigens;parasite allergens such as Ascaris antigen; viral antigens; fungalspores; drug allergens; antibiotics; penicillins and related compounds;other antibiotics; whole proteins such as hormones (insulin), enzymes(streptokinase); all drugs and their metabolites capable of acting asincomplete antigens or haptens; industrial chemicals and metabolitescapable of acting as haptens and functioning as allergens (e.g., theacid anhydrides (such as trimellitic anhydride) and the isocyanates(such as toluene diisocyanate)); occupational allergens such as flour(e.g., allergens causing Baker's asthma), castor bean, coffee bean, andindustrial chemicals described above; flea allergens; and human proteinsin non-human animals.

Allergens include but are not limited to cells, cell extracts, proteins,polypeptides, peptides, polysaccharides, polysaccharide conjugates,peptide and non-peptide mimics of polysaccharides and other molecules,small molecules, lipids, glycolipids, and carbohydrates.

Examples of specific natural, animal and plant allergens include but arenot limited to proteins specific to the following genuses: Canine (Canisfamiliaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis(Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poapratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

In a related embodiment, the present invention provides for apolynucleotide adjuvant composition and method of use where theimmunogenic composition comprises a PIKA adjuvant together with anautoimmune antigen or vaccine.

In a related embodiment, the present invention provides for animmunogenic composition and method of use, where the immunogeniccomposition comprises the PIKA adjuvant alone or together with a cancerantigen, wherein exemplary antigens include but are not limited toantigens of one or more of the cancers described in FIGS. 9A-9C.

In a related embodiment the source of the cancer antigen may be: 1)Viral proteins—for example hepatitis B virus (HBV), Epstein-Barr virus(EBV) and human papillomavirus (HPV)—are important in the development ofhepatocellular carcinoma, lymphoma, and cervical cancer, respectively;2). whole cancer cells that may be inactivated and/or nonpurified and/orsemi-purified extract of these cells; 3). tumor-associated antigens(TAAs) such as the tumor specific oncogenic proteins, glycosylatedproteins, gangliosides, glycolipide, mucins, peptide, carbohydrates andanti-idiotype monoclonal antibodies.

In a related embodiment, the use of the immunogenic compositioncomprising the polynucleotide adjuvant may be for the treatment ofcancer tumors through the prevention of further growth of existingcancers, the prevention of the recurrence of treated cancers, or theelimination of cancer cells not killed by prior treatments. Thetreatment may be administered prior to, in conjunction with, or postother therapies provided to the individual and thus may form part of anoverall combination therapy to treat the cancer.

In a related embodiment the cancer vaccine provides for therapiescapable of inducing tumor specific immune responses against both aprimary tumor and metastases. In addition, the induction of a strongimmunity may lead to the establishment of immune memory, therebyreducing or inhibiting tumor recurrence. The cancer vaccine may inducespecific antibodies against tumor-associated surface antigens andpreferably to induce cellular immune response with preferably a biastoward a Th1 immune response.

Any of a variety of known tumor-specific antigens or tumor-associatedantigens (TAA) can be included in a subject immunogenic composition. Theentire TAA may be, but need not be, used. Instead, a portion of a TAA,e.g., an epitope, may be used. Tumor-associated antigens (orepitope-containing fragments thereof) which may be used into YFVinclude, but are not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2,high molecular weight melanoma-associated antigen MAA, GD2,carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigensOV-TL3 and MOV18, TUAN, alpha-feto protein (AFP), OFP, CA-125, CA-50,CA-19-9, renal tumor-associated antigen G250, EGP-40 (also known asEpCAM), S100 (malignant melanoma-associated antigen), p53, and p21ras. Asynthetic analog of any TAA (or epitope thereof), including any of theforegoing, may be used. Furthermore, combinations of one or more TAAs(or epitopes thereof) may be included in the composition.

In some embodiments, a subject immunogenic composition comprises apolynucleotide adjuvant, and at least two different antigens, e.g., insome embodiments, a subject immunogenic composition comprises twoantigens, three antigens, four antigens, five antigens, or more thanfive antigens.

Additional Agents

In some embodiments, a subject immunogenic composition comprises, inaddition to a PIKA adjuvant and an antigen, one or more additionalagents, e.g., immunomodulatory agents, carriers, and the like.

In an embodiment of particular interest, the present invention providesfor an immunogenic composition and method of use, where the immunogeniccomposition comprises the PIKA adjuvant, an antigen or vaccine togetherwith another immunomodulating substance, including adjuvants, wheresuitable immunomodulating substances include, but are not limited to: analuminum composition such as aluminum hydroxide; oil-in-water emulsionscompositions or emulsions comprising an immunogenic substances,including Complete Freund's Adjuvant; an oil-in-water emulsioncontaining dried, heat-killed Mycobacterium tuberculosis organisms;Incomplete Freund's Adjuvant; emulsions including mycobacterial cellwall components; emulsions including squalene (MF-59); detoxifiedendotoxins including monophosphoryl lipid A-microbial (MPL); haptens;nitrocellulose-absorbed protein; saponins including particulateimmunomodulators isolated from the bark of Quillaja Saponoria forexample QS21; endogenous human immunomodulators; bacterial derivedadjuvants including unmethylated CpG dinucleotides;oligodeoxynucleotides (e.g., synthetic oligonucleotides) containingunmethylated CpG dinucleotides; liposomes (e.g., liposomes made ofbiodegradable materials such as phospholipids); biodegradable polymermicrospheres (e.g., microspheres made from a variety of polymers such aspolylactic-co-glycolic acid (PLGA), polyphosphazene and polyanhydrides);Interlukin-2; Bacillus Calmette Guerin; Granulocyte Monocyte-ColonyStimulating Factor; Montanide ISA-51; Keyhole limpet hemocyanin; DNA;proteins; encapsulated antigens; and immune stimulating complexes(ISCOM's).

In a related embodiment the present invention provides for animmunogenic substance comprising a PIKA adjuvant, an antigenic substanceor substances, plus a suitable carrier. The carrier may be for examplean oil and water emulsion, suspension, a lipid vehicle or aluminum salt.

In one embodiment of interest, the composition comprising PIKA adjuvantdoes not include poly-L-lysine or a derivative thereof.

Kits

In certain embodiments, the invention provides a kit comprising asubject immunogenic composition. In certain embodiments, the inventionprovides a kit comprising a PIKA adjuvant and an antigen in separateformulations.

In a related embodiment, the invention provides for a kit comprising thepolynucleotide adjuvant and an immunogenic compound where theimmunogenic substance is an antigen.

In some embodiments, a subject kit comprises a subject immunogeniccomposition in a sterile liquid (e.g., aqueous) formulation, where theformulation is sterile, and is provided in a sterile container, asterile vial, or a sterile syringe.

In some embodiments, a subject kit comprises a subject immunogeniccomposition formulated for injection. In some embodiments, a subject kitcomprises a subject immunogenic composition in a sterile liquidformulation, contained within a sterile syringe; and a needle. In someembodiments, a subject kit comprises a subject immunogenic compositionin a sterile liquid formulation in a unit dosage amount (e.g., a singledose), contained within a sterile syringe; and a needle.

In some embodiments, a subject kit comprises a subject immunogeniccomposition, lyophilized and in a sterile container; and a containercomprising a sterile liquid for reconstitution of the lyophilizedcomposition. In some embodiments, the kit further comprises instructionsfor reconstitution of the lyophilized composition.

A subject kit in some embodiments will further include instructions foruse, including e.g., dosage amounts and dosage frequencies. Instructionsare in some embodiments printed directly on the kit. In otherembodiments, instructions are printed material provided as a packageinsert. Instructions can also be provided in other media, e.g.,electronically in digital or analog form, e.g., on an audio cassette, anaudio tape, a compact disc, a digital versatile disk, and the like.

Formulations

A subject immunogenic composition is provided in any of a variety offormulations. For example, a subject immunogenic composition may beprepared as an injectable, dry power, liquid solution, suspension oremulsion. The preparation of formulations of a desired immunogeniccomposition is generally described in Vaccine 4^(th) Edition by StanleyA Plotkin et al., W.B. Saunders Company; 4th edition 2003. Suitableformulations are also described in, e.g., A. Gennaro (2000) “Remington:The Science and Practice of Pharmacy,” 20^(th) edition, Lippincott,Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug DeliverySystems (1999) H. C. Ansel et al., eds., 7^(th) ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.; Methodsin Molecular Medicine, Vol. 87: Vaccine Protocols, 2nd edition (2003),Humana Press; Mucosal Vaccines (1996), Kiyono et al., eds., AcademicPress; and Vaccine Adjuvants: Preparation Methods and Research Protocols(2000) D. T. O'Hagan, Humana Press.

The immunogenic composition of the present invention may be employed insuch forms, both sterile and non-sterile, such as capsules, liquidsolutions, liquid drops, emulsions, suspensions, elixirs, creams,suppositories, gels, soft capsules, sprays, inhalants, aerosols,powders, tablets, lozenges, or pills. Any inert carrier can be used,such as saline, or phosphate buffered saline, stabilizers, propellants,encased in gelatin capsule or in a microcapsule or vector that aidsadministration or any such carrier in which the compounds used in themethod of the present invention have suitable solubility properties foruse in the methods of the present invention.

In certain embodiments, the PIKA adjuvant composition and an immunogeniccomposition comprising the PIKA adjuvant and antigenic compound isfreeze-dried (lyophilized) for long term stability and storage in asolid form. The freeze-dried method is known to those skilled in theart.

In one aspect of particular interest, the invention provides for anadjuvant composition or immunogenic composition wherein the immunogeniccomposition, or the adjuvant composition contained in the immunogeniccomposition, is in a solid or liquid form or in solution or insuspension or in emulsion.

A subject immunogenic composition may be administered to an individualby means of a pharmaceutical delivery system for the inhalation route(oral, intratracheal, intranasal). Thus, a subject immunogeniccomposition may be formulated in a form suitable for administration byinhalation. The pharmaceutical delivery system is one that is suitablefor respiratory therapy by topical administration of a subject bacterialcomposition to mucosal linings of the bronchi. This invention canutilize a system that depends on the power of a compressed gas to expelthe bacteria from a container. An aerosol or pressurized package can beemployed for this purpose. As used herein, the term “aerosol” is used inits conventional sense as referring to very fine liquid or solidparticles carries by a propellant gas under pressure to a site oftherapeutic application. When a pharmaceutical aerosol is employed inthis invention, the aerosol contains the immunogenic composition, whichcan be dissolved, suspended, or emulsified in a mixture of a fluidcarrier and a propellant. The aerosol can be in the form of a solution,suspension, emulsion, powder, or semi-solid preparation. Aerosolsemployed in the present invention are intended for administration asfine, solid particles or as liquid mists via the respiratory tract of asubject. Various types of propellants known to one of skill in the artcan be utilized. Examples of suitable propellants include, but are notlimited to, hydrocarbons or other suitable gas. In the case of thepressurized aerosol, the dosage unit may be determined by providing avalue to deliver a metered amount.

There are several different types of inhalation methodologies which canbe employed in connection with the present invention. A subjectimmunogenic composition can be formulated in basically three differenttypes of formulations for inhalation. First, a subject immunogeniccomposition can be formulated with low boiling point propellants. Suchformulations are generally administered by conventional meter doseinhalers (MDI's). However, conventional MDI's can be modified so as toincrease the ability to obtain repeatable dosing by utilizing technologywhich measures the inspiratory volume and flow rate of the subject asdiscussed within U.S. Pat. Nos. 5,404,871 and 5,542,410.

Alternatively, a subject immunogenic composition can be formulated inaqueous or ethanolic solutions and delivered by conventional nebulizers.In some embodiments, such solution formulations are aerosolized usingdevices and systems such as disclosed within U.S. Pat. Nos. 5,497,763;5,544,646; 5,718,222; and 5,660,166.

Furthermore, a subject immunogenic composition can be formulated intodry powder formulations. Such formulations can be administered by simplyinhaling the dry powder formulation after creating an aerosol mist ofthe powder. Technology for carrying such out is described within U.S.Pat. No. 5,775,320 and U.S. Pat. No. 5,740,794.

Formulations suitable for intranasal administration include nasalsprays, nasal drops, aerosol formulations; and the like.

In some embodiments, a subject immunogenic composition is formulated asa sustained release (e.g. a controlled release formulation). Forexample, in some embodiments, a subject immunogenic composition isformulated into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants. Such implants willgenerally employ known inert materials such as biodegradable polymers.Injectable depot forms are made by forming microencapsule matrices of asubject immunogenic composition in biodegradable polymers such aspolylactide-polyglycolide. Examples of other suitable biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the compositionin liposomes or microemulsions which are compatible with body tissue.

Methods

In one aspect of particular interest, the invention provides for amethod for eliciting and/or enhancing immune responses to an antigeniccompound, comprising administering to a host a subject immunogeniccomposition. In some embodiments, the host is a human. In otherembodiments, the host is a non-human animal, e.g., a non-human mammal,an avian species, etc.

In certain embodiments, the polynucleotide adjuvant composition can beused in the context of a vaccine. Optionally, the vaccine compositioncontains additional adjuvants. Vaccines classes included areanti-infectious diseases, anti cancer, anti-allergy and anti-auto-immunediseases.

Furthermore, the present invention provides a method for enhancingimmune responses to an antigenic compound by administering to a host asubject immunogenic composition. The host can be a human being ornon-human animal.

A subject immunogenic composition is in some embodiments deliveredparenterally by injection, such as intramuscular, intraperitoneal,intravenous, subcutaneous or intradermal injection. In other embodimentsthe immunogenic composition is administered intradermally in ways otherthan by injection. for example, without breaching the epithelial barrierby mechanical means. In other embodiments, the immunogenic compositionis delivered rectally, vaginally, nasally, orally (includinginhalation), opthamalically, topically, pulmonary or transdermally.

In certain embodiments, when the mode of administration of theimmunogenic composition comprising a polynucleotide adjuvant is for thetreatment of cancer tumors, the delivery is by injection directly intothe tumor, or adjacent to the tumor. In some embodiments, theimmunogenic composition is delivered evenly over or throughout the tumorto enhance the biodistribution and hence enhance the therapeuticbenefit.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. Exemplary injection media which canbe used in the present invention include a buffer with or withoutdispersing agents and/or preservatives, and edible oil, mineral oil, codliver oil, squalene, mono-, di- or triglyceride, and a mixture thereof.

A subject immunogenic composition is administered in an “effectiveamount” that is, an amount of a subject immunogenic composition that iseffective in a selected route of administration to elicit, induce, orenhance an immune response. In some embodiments, an immune response iselicited to antigens produced by a pathogenic microorganism. In someembodiments, the amount of a subject immunogenic composition iseffective to limit an infection, and/or to eradicate an infection,and/or to reduce a symptom associated with infection, by a pathogenicorganism.

For example, in some embodiments, administration of a subjectimmunogenic composition to an individual is effective to treat aninfectious disease, where treating an infectious disease, encompassesone or more of reducing the number of pathogenic agents in theindividual (e.g., reducing viral load, reducing bacterial load, reducingthe number of protozoa, reducing the number of helminths) and/orreducing a parameter associated with the infectious disease, including,but not limited to, reduction of a level of a product produced by theinfectious agent (e.g., a toxin, an antigen, and the like); and reducingan undesired physiological response to the infectious agent (e.g.,fever, tissue edema, and the like).

The exact amount of such compositions required will vary from subject tosubject, depending on the species, age, weight, and general conditionsof the subject, the severity of the disease, infection, or conditionthat is being treated or prevented, the particular compound used, itsmode administration, and the like. An appropriate amount may bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein. Following an initialadministration, subjects may receive one or several boosterimmunizations adequately spaced.

In some embodiments, serial doses of a subject immunogenic compositionare administered. In these embodiments, after administration of a firstdose of a subject immunogenic composition, a second dose of a subjectimmunogenic composition is administered to the individual after theindividual has been immunologically primed by exposure to the firstdose. The booster may be administered days, weeks or months after theinitial immunization, depending upon the patient's response andcondition. For example, the booster dose is administered from about 2days to about 12 months after the initial dose, e.g., from about 2 daysto about 7 days, from about 1 week to about 2 weeks, from about 2 weeksto about 4 weeks, from about 4 weeks to about 8 weeks, from about 8weeks to about 6 months, or from about 6 months to about 12 months afterthe initial dose. The present invention further contemplates the use ofa third, fourth, fifth, sixth or subsequent booster immunization, using,e.g., a third, fourth, fifth, sixth, or subsequent dose.

Whether an antibody response to an antigen has been induced or enhancedin an individual is readily determined using standard assays. Forexample, immunological assays such as enzyme-linked immunosorbent assays(ELISA), radioimmunoassay (RIA), immunoprecipitation assays, and proteinblot (“Western” blot) assays; and neutralization assays (e.g.,neutralization of viral infectivity in an in vitro or in vivo assay);can be used to detect the presence of antibody specific for a microbialantigen in a bodily fluid or other biological sample, e.g., the serum,secretion, or other fluid, of an individual.

Whether a CD4 immune response to an antigen has been induced in anindividual is readily determined using standard assays, e.g.,fluorescence-activated cell sorting (FACS) (see, e.g., Waldrop et al.(1997) J. Clin. Invest. 99:1739-1750); intracellular cytokine assaysthat detect production of cytokines following antigen stimulation (see,e.g., Suni et al. (1998) J. Immunol. Methods 212:89-98; Nomura et al.(2000) Cytometry 40:60-68; Ghanekar et al. (2001) Clin. Diagnostic Lab.Immunol. 8:628-631); MHC-peptide multimer staining assays, e.g., use ofdetectably labeled (e.g., fluorescently labeled) soluble MHC ClassII/peptide multimers (see, e.g., Bill and Kotzin (2002) Arthritis Res.4:261-265; Altman et al. (1996) Science 274:94-96; and Murali-Krishna etal. (1998) Immunity 8:177-187); enzyme-linked immunospot (ELISPOT)assays (see, e.g., Hutchings et al. (1989) J. Immunol. Methods 120:1-8;and Czerkinsky et al. (1983) J. Immunol. Methods 65:109-121); and thelike. As one non-limiting example of an intracellular cytokine assay,whole blood is stimulated with antigen and co-stimulating antibodies(e.g., anti-CD28, anti-CD49d) for 2 hours or more; Brefeldin A is addedto inhibit cytokine secretion; and the cells are processed for FACSanalysis, using fluorescently labeled antibodies to CD4 and to cytokinessuch as TNF-α, IFN-γ and IL-2.

Whether an antigen-specific CD8 (e.g., cytotoxic T cell; “CTL”) responseis induced to an antigen (e.g., to a pathogen) can be determined usingany of a number of assays known in the art, including, but not limitedto, measuring specific lysis by CTL of target cells expressing theantigen on their surface, which target cells have incorporated adetectable label which is released from target cells upon lysis, and canbe measured, using, e.g., a ⁵¹Cr-release assay; a lanthanidefluorescence-based cytolysis assay; and the like.

Subjects Suitable for Treatment

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orpreventing an infection with a microbial pathogen, include individualswho have been infected with a pathogenic microorganism; individuals whoare susceptible to infection by a pathogenic microorganism, but who havenot yet been infected; and individuals who are at risk of becominginfected with a pathogenic microorganism, but who have not yet beeninfected. Suitable subjects include infants, children, adolescents, andadults.

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include pediatrictarget population, e.g., individuals between about 1 year of age andabout 17 years of age, including infants (e.g., from about 1 month oldto about 1 year old); children (e.g., from about 1 year old to about 12years old); and adolescents (e.g., from about 13 years old to about 17years old).

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include neonates, e.g.,an individual (e.g., a human neonate) from one day to about 14 days old,e.g., from about 1 day to about 2 days old, from about two days to about10 days old, or from about 10 days to about 14 days old.

In a particular embodiment, the subject is a human child about ten yearsor younger, e.g., about five years old or younger, and the immunogeniccompositions are administered at any one or more of the following times:two weeks, one month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15months, 18 months, or 21 months after birth, or at 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years of age.In some embodiments, a subject immunogenic composition is administeredto an individual in the age range of from about 6 months to about 6years, where the individual receives a first dose at about 6 months ofage, and subsequent booster doses, e.g., 2-3 subsequent booster doses,at, e.g., 2 years of age, 4 years of age, and 6 years of age.

In some embodiments, a subject immunogenic composition is administeredto an individual shortly after contact (e.g., shortly after confirmed orsuspected contact) with an actual or potential source of the microbialpathogen, for example, an individual who is known to have or suspectedto have an infection with a microbial pathogen. For example, in someembodiments, a subject immunogenic composition is administered to anindividual within about 1 hour, within about 2 hours, within about 5hours, within about 8 hours, within about 12 hours, within about 18hours, within about 24 hours, within about 2 days, within about 4 days,within about 7 days, within about 2 weeks, or within about one monthafter contact with an individual who is known to have or suspected tohave an infection with a microbial pathogen.

In some embodiments, a subject immunogenic composition is administeredto an individual that is known or may be suspected of being a carrier ora microbial pathogen whether or not they are showing symptoms of theinfection.

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include CD4⁺ Tcell-deficient individuals (“CD4⁺-deficient” individuals), e.g.,individuals who have lower than normal numbers of functional CD4⁺ Tlymphocytes. As used herein, the term “normal individual” refers to anindividual having CD4⁺ T lymphocyte levels and function(s) within thenormal range in the population, for humans, typically 600 to 1500 CD4⁺ Tlymphocytes per mm³ blood. CD4⁺-deficient individuals includeindividuals who have an acquired immunodeficiency, or a primaryimmunodeficiency. An acquired immunodeficiency may be a temporary CD4⁺deficiency, such as one caused by radiation therapy, or chemotherapy.

Also suitable for treatment with the methods of the invention areindividuals with healthy, intact immune systems, but who are at risk forbecoming CD4⁺ deficient (“at-risk” individuals). At-risk individualsinclude, but are not limited to, individuals who have a greaterlikelihood than the general population of becoming CD4⁺ deficient.Individuals at risk for becoming CD4⁺ deficient include, but are notlimited to, individuals at risk for HIV infection due to sexual activitywith HIV-infected individuals; intravenous drug users; individuals whomay have been exposed to HIV-infected blood, blood products, or otherHIV-contaminated body fluids; a baby who has passed through the birthcanal of an HIV-infected individual; babies who are being nursed byHIV-infected mothers; and the like.

Subjects suitable for treatment with a subject method for treatingcancer include individuals who have been diagnosed with cancer;individuals who were previously treated for cancer, e.g., bychemotherapy or radiotherapy, and who are being monitored for recurrenceof the cancer for which they were previously treated; and individualswho have undergone bone marrow transplantation or any other organtransplantation.

Subjects suitable for treatment with the formulations and methods of theinstant invention for treating allergy include any individual who hasbeen diagnosed as having an allergy. Subjects amenable to treatmentusing the methods and agents described herein include individuals whoare known to have allergic hypersensitivity to one or more allergens.Subjects amenable to treatment include those who have any of theabove-mentioned allergic disorders. Also amenable to treatment aresubjects that are at risk of having an allergic reaction to one or moreallergens. Also suitable are individuals who failed treatment with oneor more standard therapies for treating an allergic disorder.

Subjects suitable for treatment include individuals living inindustrialized nations; individuals living developing countries;individuals living in rural areas; individuals living in relativelyisolated areas; and the like.

The target population for a subject immunogenic composition will vary,depending on the microbial pathogen

The above disclosure generally describes the present invention. Thefollowing examples will be of assistance to the understanding of thepresent invention. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES Example 1 PIKA in Combination with a Variety of AntigensInduces a Specific Immune Response

This example involves use of PIKA in combination with a variety ofantigens to elicit a specific immune response in vivo. The research wasconducted in a series of independent experiments with a common protocolthough using a different antigen each time. The antigens tested include:a recombinant protein hepatitis B surface antigen type adw, aninactivated split influenza vaccine (VAXIGRIP from Sanofi Pasteur), asynthesized HIV peptide antigen, a recombinant protein herpes simplexvirus type 2 gD antigen, recombinant protective anthrax protein antigen,inactivated whole virus avian influenza antigen strain H5N1 and aninactivated whole virus Severe Acute Respiratory Syndrome (SARS)inactivated antigen.

The protocol for the individual experiment involve the inoculation ofBalb/c mice with compositions of antigen alone, antigen with the PIKAadjuvant (a heterogeneous composition of PIKA molecules predominantlywithin a weight range distribution of about 66 kDa to 1,200 kDa), PIKAalone, a control comprising phosphate buffer solution (PBS). The groupsare designated A to E according to the table I below: TABLE IComposition Definition Group Composition A Antigen only  B* Antigen andPIKA adjuvant C PIKA only D Control (phosphate buffer solution)*Different ratios/quantities of antigen and adjuvant are designated B1,B2 etc.

Actual dosage quantities are provided for each antigen used. The micewere then given an identical booster vaccine ten to fourteen days afterthe initial injection. Ten to fourteen days after the booster injectiona blood sample was taken, the mice were then sacrificed and tissuesamples taken from the spleen.

A suspension of spleen cells was prepared and a sample of the cellsuspension from each mouse was put into 6-12 wells of the ELISPOT plateand cultured, Each well of the ELISPOT plate contained 200 ul ofsplenocyte suspension, equivalent to approximately 2×10⁵ or 2.5×10⁵cells/well (see units details in tables below). For each mouse's sampleof cultured splenocytes, half of wells containing the splenocytes wereincubated with culture medium and the other half of wells werestimulated using the one of two different concentrations of particularantigen under evaluation. Plates are incubated at 37° C. for 20 hours inenvironmentally controlled conditions prior to final preparation andreading using a standard ELISPOT plate reader.

Standard ELISPOT tests, known to those skilled in the art, were used todetect the number of cells producing the cytokines IL-4, IL-2 and INF-γ.

Flow Cytometry analysis was used to detect INF-γproduced by CD4+ andCD8+ T cells. The use of Fluorescence-Activated Cell Sorter (FACS) iswell known by those skilled in the art. In brief solutions ofsplenocytes at a concentration of 2.5×10⁶ cells/ml were prepared anddivided into individual tubes with 2 ml per sample. Samples stimulatedwith antigen were then prepared and after incubation at 37C inenvironmentally controlled conditions for 5 hours. The samples then werewashed and stained prior to reading in a standard FACS reader.

The standard ELISA tests for the presence of antibodies in blood serumis well known to persons skilled in the art.

Example 1.1 Recombinant Hepatitis B Surface Antigen (HBSAg) adw

The results in table II below are the results of the ELISPOT testdetecting the presence number of cells producing INF-γ, IL-2 and IL-4using a recombinant protein hepatitis B surface antigen (HBsAg) typeadw. Groups A to E represent the different combinations ofantigen/adjuvant/control media administered to the mice. The numbers inthe table II (see also FIG. 1.1) represent the ELISPOT reading, thenumber of spot forming cells, that is, a direct measure the number ofcells producing cytokine.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to recombinanthepatitis B surface antigen enhances the expression of cytokines INF-γ,IL-2 and IL-4 by cultured spleen cells. The observed expression ofcytokines indicates an enhanced adaptive immune response of both ahumoral and cell mediated immunity induced by the presence of the PIKAadjuvant. TABLE II Expression of Cytokines by Mice Spleen Cells MiceGroups A B1 B2 C D Immunized with HBsAg 4 ug  4 ug  4 ug (ug/mouse/inj.)PIKA 50 ug 200 ug 100 ug PBS No of Cell producing IFN-γ/ non stimulated3 3 2 3 2 2 × 10⁵ splenocyte stimulated w/ 15 41 114 2 1 0.5 ug/mlstimulated w/ 21 101 214 1 1 5 ug/ml No of Cell producing IL_2/ nonstimulated 2 0 1 1 1 2 × 1010⁵ splenocyte stimulated w/ 12 9 124 2 1 0.5ug/ml stimulated w/ 42 47 197 1 1 5 ug/ml No of Cell producingIL-4/ nonstimulated 10 9 8 1 1 2 × 10⁵ stimulated w/ 37 27 21 2 1 splenocyte 0.5ug/ml stimulated w/ 57 41 40 1 0 5 ug/mlug = micrograms

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to HBsAg, in particular the specific immuneresponse, more particularly the adaptive immunity and more specificallypredominant Th1 bias immune response and promote the cell mediatedimmune response.

Example 1.2 VAXIGRIP (Sanofi Pasteur), Inactivated and PurifiedInfluenza Antigen Comprising; H1N1, H3N2 Like Strains andb/Shanghai5/361/2002 Strain

The results in table III below are the results of the ELISPOT testdetecting the presence the number of cells producing INF-γ, IL-2 andIL-4 using VAXIGRIP vaccine a inactivated split human influenza vaccineproduced by Sanofi Pasteur. Groups A to D represent the differentcombinations of antigen/adjuvant/control media administered to the mice.The numbers in the table III (see also FIG. 1.2) represent the ELISPOTreading, the number of spot forming cells, that is, a direct measure ofcytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the influenzaantigen enhances the expression of cytokines INF-γ, IL-2 and IL-4 bycultured spleen cells. The observed expression of cytokines indicates anenhanced adaptive immune response of both a humoral and cell mediatedimmunity induced by the presence of the PIKA adjuvant. TABLE IIIExpression of Cytokines by Mice Spleen Cells Mice Groups A B1 B2 C DImmunized with Vaxigrip 4.5 ug 4.5 ug   4.5 ug (ug/mouse/inj.) PIKA 50ug 100 ug 100 ug PBS No of Cell producing IFN-γ/ non stimulated 1 1 2 20 2 × 10⁵ splenocyte stimulated w/ 81 148 252 14 6 0.5 ug/ml stimulatedw/ 48 122 233 1 4 5 ug/ml No of Cell producing IL-2/ non stimulated 1 11 1 0 2 × 10⁵ splenocyte stimulated w/ 23 85 122 1 1 0.5 ug/mlstimulated w/ 33 97 139 1 1 5 ug/ml No of Cell producing IL-4/ nonstimulated 7 11 8 8 7 2 × 10⁵ splenocyte stimulated w/ 25 38 51 9 9 0.5ug/ml stimulated w/ 37 34 33 5 10 5 ug/ml

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to influenza antigen, in particular thespecific immune response, more particularly the adaptive immunity andmore specifically the cell mediated immune response.

The VAXIGRIP is an approved influenza vaccine recognized tosignificantly reduce the risk of contracting influenza. The addition ofPIKA enhances the level of cytokines produced thereby indicating that avaccine comprising VAXIGRIP and PIKA also elicits an immune responsethat significantly reduces the risk of contracting influenza.

Example 1.3 Synthesized HIV Peptide Antigen

The results in table IV below are the results of the ELISPOT testdetecting the presence number of cells producing INF-γ, IL-2 and IL-4using an HIV peptide antigen. Groups A to D represent the differentcombinations of antigen/adjuvant/control media administered to the mice.The numbers in the table IV (see also FIG. 1.3 a) represent the ELISPOTreading, the number of spot forming cells, that is, a direct measure ofcytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the HIV antigenenhances the expression of cytokines INF-γ, IL-2 and IL-4 by culturedspleen cells. The observed expression of cytokines indicates an enhancedadaptive immune response of both a humoral and cell mediated immunityinduced by the presence of the PIKA adjuvant. TABLE IV Expression ofCytokines by Mice Spleen Cells Mice Groups A B C D Immunized with HIVpeptide 2.0 ug  2.0 ug (ug/mouse/inj.) PIKA 100 ug 100 ug PBS No of Cellnon stimulated 1 1 2 1 producing IFN-γ/ stimulated w/ 16 121 2 5 2.5 ×10⁵ 2.5 ug/ml Splenocyte No of Cell non stimulated 3 1 2 0 producingIL-2/ stimulated w/ 56 166 4 5 2.5 × 10⁵ 2.5 ug/ml Splenocyte No of Cellnon stimulated 5 11 5 3 producing IL-4/ stimulated w/ 78 68 5 6 2.5 ×10⁵ 2.5 ug/ml Splenocyte

The results of the FACS analysis are presented in table V below (seealso FIG. 1.3 b). The presence of CD4+ T cells expressing INF-γin onlythe formulations containing both PIKA and HIV antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process. TABLE V HIVCD4+ T Cell FACS Analysis Group A B1 B2 C D HIV Antigen 2 ug  2 ug  2 ug— — PIKA — 100 ug 100 ug 100 ug PBS Percentage CD4 Positive CellsExpressing INF-γ Sample 0.02% 0.19% 0.15% 0.01% 0.01% Control 0.00%0.01% 0.00% 0.01% 0.01%

The conclusion drawn is that the addition of the PIKA adjuvant with HIVantigen enhances the overall immune response, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 1.4 Recombinant Anthrax Protective Antigen from BacillusAnthracis

The results in table VI below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using a recombinantanthrax. Groups A to D represent the different combinations ofantigen/adjuvant/control media administered to the mice. The numbers inthe table VI (see also FIG. 1.4 a) represent the ELISPOT reading, thenumber of spot forming cells, that is, a direct measure of cytokineproduction.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the anthraxantigen enhances the expression of cytokines INF-γ, IL-2 and IL-4 bycultured spleen cells. The observed expression of cytokines indicates anenhanced adaptive immune response of both a humoral and cell mediatedimmunity induced by the presence of the PIKA adjuvant. TABLE VIExpression of Cytokines by Mice Spleen Cells Mice Groups A B C DImmunized with Anthrax rPA 3.0 ug  3.0 ug (ug/mouse/inj.) PIKA 100 ug100 ug PBS No of Cell non stimulated 1 1 2 1 producing IFN-γ/ stimulatedw/ 16 121 2 5 2.5 × 10⁵ 2.5 ug/ml Splenocyte No of Cell non stimulated 247 2 0 producing IL-2/ stimulated w/ 12 134 3 4 2.5 × 10⁵ 2.5 ug/mlSplenocyte No of Cell non stimulated 4 9 5 3 producing IL-4/ stimulatedw/ 10 61 6 3 2.5 × 10⁵ 2.5 ug/ml Splenocyte

The results of the FACS analysis are presented in table VII below (seealso FIG. 1.4 b). The presence of CD4+ T cells expressing INF-γ in onlythe formulations containing both PIKA and rPA antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process. TABLE VIIAnthrax CD4+ T Cell FACS Analysis Group A B1 B2 C D Anth. Antigen 3 ug 3 ug  3 ug — — PIKA — 100 ug 100 ug 100 ug PBS Percentage CD4 PositiveCells Expressing INF-γ Sample 0.01% 0.75% 0.48% 0.01% 0.00% Control0.00% 0.01% 0.00% 0.01% 0.00%

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to rPA, in particular the specific immuneresponse, more particularly the adaptive immunity and more specificallythe cell mediated immune response.

Example 1.5 Recombinant Herpes Simplex Virus 2 gD Antigen

The results in table VIII below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using a recombinantherpes simplex virus antigen. Groups A to D represent the differentcombinations of antigen/adjuvant/control media administered to the mice.The numbers in the table VIII (see also FIG. 1.5 a) represent theELISPOT reading, the number of spot forming cells, that is, a directmeasure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the herpessimplex virus antigen enhances the expression of cytokines INF-γ, IL-2and IL-4 by cultured spleen cells. The observed expression of cytokinesindicates an enhanced adaptive immune response of both a humoral andcell mediated immunity induced by the presence of the PIKA adjuvant.TABLE VIII Expression of Cytokines by Mice Spleen Cells Mice Groups A BC D Immunized with HSV2 rPA 3.0 ug  3.0 ug (ug/mouse/inj.) PIKA 100 ug100 ug PBS No of Cell non stimulated 2 2 0 1 producing IFN-γ/ stimulatedw/ 135 266 2 12 2.5 × 10⁵ 2.5 ug/ml Splenocyte No of Cell non stimulated2 2 0 0 producing IL-2/ stimulated w/ 57 153 4 4 2.5 × 10⁵ 2.5 ug/mlSplenocyte No of Cell non stimulated 13 17 11 17 producing IL-4/stimulated w/ 25 40 10 12 2.5 × 10⁵ 2.5 ug/ml Splenocyte

The results of the FACS analysis are presented in table IX below (seealso FIG. 1.5 b). The presence of CD4+ T cells expressing INF-γin onlythe formulations containing both PIKA and HSV antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process. TABLE IX HerpesSimples Virus CD4+ T Cell FACS Analysis Group A B1 B2 C D HSV Antigen 3ug  3 ug  3 ug — — PIKA — 100 ug 100 ug 100 ug PBS Percentage CD4Positive Cells Expressing INF-γ Sample 0.12% 0.35% 1.21% 0.02% 0.02%Control 0.01% 0.02% 0.00% 0.01% 0.00%

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to HSV antigen, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 1.6 Inactivated H5N1 Whole Virus (Avian Influenza) Antigen

The results in table X below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using an inactivated nonpurified H5N1 antigen. Groups A to D represent the differentcombinations of antigen/adjuvant/control media administered to the mice.The numbers in table X (see also FIG. 1.6 a) represent the ELISPOTreading, the number of spot forming cells, that is, a direct measure ofcytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the H5N1 antigenenhances the expression of cytokines INF-γ, IL-2 and IL-4 by culturedspleen cells. The observed expression of cytokines indicates an enhancedadaptive immune response of both a humoral and cell mediated immunityinduced by the presence of the PIKA adjuvant. TABLE X Expression ofCytokines by Mice Spleen Cells Mice Groups A B C D Immunized with H5N1100 ul  100 ul (ul or ug/mouse/inj.) PIKA 100 ug 100 ug PBS No of Cellnon stimulated 2 2 0 0 producing IL-2/ stimulated w/ 97 135 1 2 2.5 ×10⁵ 14 ul/well Splenocyte No of Cell non stimulated 11 9 11 17 producingIL-4/ stimulated w/ 137 184 10 9 2.5 × 10⁵ 14 ug/well Splenocyte

The results of the FACS analysis are presented in table XI below (seealso FIG. 1.6 b and c). The presence of CD4+ and CD8+ T cells expressingINF-γin only the formulations containing both PIKA and H5N1 antigenconfirms the observation that the adaptive immune response has reach astage of maturity and that PIKA was instrumental in this process. TABLEXI H5N1 CD4+ and CD8+T Cell FACS Analysis Group A B1 B2 C D H5N1 Antigen100 ul  100 ul  100 ul  — — PIKA — 100 ug 100 ug 100 ug PBS PercentageCD4 Positive Cells Expressing INF-γ Sample 0.75% 2.13% 2.44% 0.01% 0.02%Control 0.00% 0.00% 0.01% 0.01% 0.01% Percentage CD8 Positive CellsExpressing INF-γ Sample 0.08% 0.16% 0.26% 0.04% 0.01% Control 0.03%0.01% 0.02% 0.05% 0.01%

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to H5N1 antigen, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 2 Inactivated Whole Virus SARS Antigen

The objective of this experiment is to demonstrate that the addition ofPIKA to a SARS antigen enhances the immune response and stimulates thehost's immune system to produce protective SARS specific antibodies.

In this program of research six groups each comprising 4 Balb/c micewere inoculated (peritoneal injection) with a combination of SARSantigen, the antigen plus PIKA (a heterogeneous composition of PIKAmolecules predominantly within a weight range of 66 kDa to 1,200,000kDa), PIKA alone or a control, see table XII below (see also FIG. 2).

Each group was administered an identical doses on day 0, day 14 and day28. On week six a blood sample was extracted and the serum tested forthe presence of IgG, being a measure of the presence of disease specificantibodies. The blood serum was diluted by a factor of 16,000 times thenthe presence of IgG was measured using an ELISA reader the procedurebeing familiar to those skilled in the art. The output being an opticaldensity (O.D.) reading where the greater the value the greater thepresence of IgG.

The results, presented in table XII, demonstrated a correlation betweenthe presence of the PIKA adjuvant and an increase in the expression ofIgG. TABLE XII Optical Density Readings of IgG Antibody Using ELISAGroup A B1 B2 C D SARS Antigen 10 ug 10 ug  10 ug PIKA 50 ug 100 ug 100ug PBS O.D. Reading 0.26 0.41 0.40 0.09 0.09 Relative value 1.0 1.6 1.60.3 0.3

The conclusion is that the presence of PIKA with the SARS antigenincreases the expression of IgG in a dose dependent manner therebyenhancing the immune response of the host.

Example 3 PIKA Vaccine Provides Immune Protection Against H5N1 Infection

The objective of this experiment is to demonstrate that an avianinfluenza vaccine comprising the PIKA adjuvant is able to protectchickens against live avian flu virus infection.

The research was conducted on two groups of 24 SPF chickens each. At tendays old the birds were inoculated subcutaneously in the neck with a 700ul dose of vaccine comprising PIKA (a heterogeneous composition of PIKAmolecules predominantly within a weight range of 66 kDa to 660 kDa) andtwo strains of avian influenza (H5N1 and H9N2). The composition includedantigen and PIKA adjuvant at a ratio of approximately 2:1 antigen to thePIKA adjuvant.

Blood samples were taken from under the wing at 7, 14 and 21 days. Theblood serum was tested for the presence of specific H5 and H9antibodies.

At 21 days the birds were challenged with the H5N1 live virus and thenobserved for an additional 14 days. The survival rate of the chickensafter the 14 days exposure to the live H5N1 virus was recorded.

Table XIII (see also FIG. 3) demonstrates that the presence of PIKAinduces the production of specific antigen antibodies. TABLE XIIIAntibody Levels Measured by ELISA Reader Antibody Day 0 Day 7 Day 14 Day21 H5 0 1.2 1.44 2.4 H9 0 1.7 2.3 3.9Units:Optical density reading from ELISA analysis

Of the 24 chickens that were vaccinated with the antigen/PIKAcomposition 21 (83%) survived for 14 days after exposure to the liveH5N1 virus. In the control group of 24 chickens that received no vaccinebut were also exposed to the live H5N1 virus only 4 (17%) were aliveafter 14 days.

The conclusion drawn is that the PIKA vaccine confers a significantlevel of immune protection against the H5N1 virus.

Example 4 PIKA Vaccine Provides Immune Protection Against RabiesInfection

The objective of this research is to demonstrate that a rabies vaccinecomprising the PIKA adjuvant is able to confer protection against arabies infection.

Four groups (designated i, ii, iii and iv) of 20 Balb/c SPF Kunming micewere each challenged with 100 ul of wild rabies virus strain CQ92. Eachgroup receive inoculations of different types of vaccine; i) acomposition of PIKA (a heterogeneous composition of PIKA moleculespredominantly within a weight range of 66 kDa to 660 kDa) andinactivated purified hamster kidney cell rabies antigen in a ratio of1:4 by volume, ii) Sanofi-Aventis' Veroab vero cell inactivated rabies,vaccine iii) the inactivated purified hamster kidney cell rabies vaccinewith an alum adjuvant and iv) control phosphate buffer solution. A 60 uldose of vaccine was administered 30 to 40 minutes, 3 days, 6 days and 9days after infection by subcutaneous injection into the thigh.

The survival rate of each groups presented in table XIV. (see also FIG.4). TABLE XIV Survival Rates Survival Vaccine Mice Number % PIKAcomposition 20 16 80% Aventis 20 2 10% Chinese 20 3 15% Control (PBS) 204 20%

The conclusion drawn is that the presence of PIKA significantly enhancesthe immune protection provided by the inactivated purified hamsterkidney cell rabies antigen.

1. An immunogenic composition comprising a PIKA adjuvant and an antigen,wherein the immunogenic composition is formulated for sustained release.2. An immunogenic composition comprising PIKA adjuvant and a viralantigen wherein the antigen is an antigen of adeniviridae, arenaviridae,astroviridae, bunyaviridae, cliciviridae, flaviviridae, hepatitis deltavirus, hepeviridae, mononegavirales, nidovirales, piconaviridae,orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae,poxyiridae, reoviridae, retroviridae or togaviridae.
 3. An immunogeniccomposition comprising PIKA adjuvant and a bacterial antigen wherein theantigen is an antigen of actinobacteria, chlamydiae, firmicutes,proteobacteria or spirochaetes.
 4. An immunogenic composition comprisingPIKA adjuvant and a fungal antigen wherein the antigen is an antigen ofascomycotaor or basidiomycota.
 5. An immunogenic composition comprisingPIKA adjuvant and a parasitic antigen wherein the antigen is an antigenof phylum sarcomastigophora, phylum apiicomplexa, phylum ciliophora,phylum plathyhelminthes, phylum nematoda or phylum arthropoda.
 6. Animmunogenic composition comprising PIKA adjuvant and a cancer antigenwherein the antigen is a cancer antigen of bone, brain, breast,digestive/gastrointestinal, endocrine, eye, genitourinary, germ cell,gynecologic, head and neck, hematological/blood, lung, musculoskeletal,neurologic, respiratory/thoracic or skin cancer.
 7. An immunogeniccomposition comprising PIKA adjuvant and a combination of two or moreantigens wherein the antigens are antigens of adeniviridae,arenaviridae, astroviridae, bunyaviridae, cliciviridae, flaviviridae,hepatitis delta virus, hepeviridae, mononegavirales, nidovirales,piconaviridae, orthomyxoviridae, papillomaviridae, parvoviridae,polyomaviridae, poxyiridae, reoviridae, retroviridae, togaviridae,actinobacteria, chlamydiae, firmicutes, proteobacteria or spirochaetes,ascomycotaor basidiomycota, phylum sarcomastigophora, phylumapiicomplexa, phylum ciliophora, phylum plathyhelminthes, phylumnematoda or phylum arthropoda.
 8. An immunogenic composition comprisingclaims 1 to 7 plus an immunomodulator
 9. An immunogenic composition ofany of claims 1 to 7 where the antigen is an inactivated microorganism,attenuated microorganism, recombinant polypeptide or syntheticpolypeptide.
 10. The immunogenic composition of any of claims 1 to 7wherein the immunogenic composition or the PIKA adjuvant contained inthe immunogenic composition, is in the form of a liquid, liquidsolution, liquid drops, a solid, capsules, emulsions, suspensions,elixirs, creams, suppositories, gels, soft capsules, sprays, inhalants,aerosols, tablets, powders, tablets or lozenges.
 11. The immunogeniccomposition of any of claims 1 to 7 wherein at least one of the adjuvantcomposition or the immunogenic composition is freeze-dried.
 12. A kitcomprising the immunogenic composition of any of claims 1 to
 7. 13. Amethod for enhancing an immune response in a host to an antigen, themethod comprising administering to the host the immunogenic compositionof any of claims 1 to
 7. 14. The method of claim 13, wherein saidadministering is by parenteral injection, intramuscular injection,intraperitoneal injection, intravenous injection, subcutaneousinjection, topical delivery, transdermal delivery or intradermaldelivery.
 15. The use of the immunogenic composition of any of claim 1to 7 for the preparation of a medicament for enhancing the immuneresponse of a host to the antigen.
 16. The method of claim 13, whereinthe host is human.
 17. The method of claim 13, wherein the host is anon-human animal.